Purification and characterization of hepatic triacylglycerol lipase isolated from rainbow trout,Oncorhynchus mykiss

Harmon, Jamie S.; Michelsen, Kim; Sheridan, Mark A.

doi:10.1007/bf02265156

Cited by 20 publications

(6 citation statements)

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“…Protein bands were purified and reduced to a single homogenate band with 70 kDa molecular weight in ion exchange chromatography, DEAE-Cellulose. Other studies found the molecular weight as 74 kDa for liver lipase of C. carpio [14], 87 kDa for lipase from the dorsal part of Cirrhinus reba [15], 40-43 kDa for O. mykiss liver lipase [20], 43 kDa for S. aurita digestive lipase [27]. As can be seen from these results, we may conclude that lipases have different molecular weight depending on the tissue and the fish species under investigation.…”

Section: Sds-pagesupporting

confidence: 53%

“…Optimum temperature was found to be 15°C for O. mykiss liver lipase [18], 37°C in Cyprinuscarpio [14], 25°C to 30° C for Gadusmorhua digestive lipase [16] and 35°C for C. reba dorsal part lipase [15].…”

Section: Physical Characterizationmentioning

confidence: 99%

“…K m and V max values for Cyprinus carpio [14] liver lipase were 0.17 mM and 2.6 μmol/ml.dk using p-NPB as substrate respectively. Harmon et al [20] found the K m and V max values as 0.28 mM and 0.016 nmol/h/mg, using tributyrin for O. mykiss liver lipase. Another study focused on liver lipase of O. mykiss determined K m and V max values as 0.12 mM and 0.40 U/mg, respectively, using p-Nitrophenyl acetate (p-NPA) as substrate [21].…”

Section: Kinetic Studymentioning

confidence: 99%

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Isolation, Purification and Characterization of Secondary Structure and Kinetic Study of Lipase from Indian Major Carp, Catla catla (Catla)

Tyagi¹

2014

Enz Eng

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Lipases are ubiquitous enzymes which catalyze the hydrolysis of fats into fatty acids and glycerol at the waterlipid interface and reversing the reaction in non-aqueous media. Lipases occupy a place of prominence among biocatalysts owing to their novel and multifold applications in oleochemistry, organic synthesis, detergent formulation and nutrition. Lipase was extracted and isolated from the alimentary canal and digestive gut of Catla catla (catla). The tissue was homogenized in a ratio of 1:3 with starting buffer (0.01 M TrisHCl, pH 7.2). The crude extract thus obtained was precipitated using ammonium sulphate (20-80%). Excess salt was removed by Dialysis and the resultant dialysate (Desalted Enzyme) was subjected to DEAE-Cellulose column for Ion Exchange Chromatography at a flow rate of 0.5 ml/min. Elution was carried out by a step gradient of NaCl (100-800 mM) in the starting buffer. Active fractions were pooled as Purified Fraction (PF) and were used for physical characterization of pH, temperature and effect of calcium on the enzyme activity, structural characterization, molecular characterization and for kinetic studies. The Purified Fraction (PF) showed final specific activity of 1438.72 U/mg. The optimum pH was 7.8 and the optimum temperature was found to be 20˚C. Melting Temperature (T m) value was 42˚C and the activation energy of purified lipase was 34.82 KJ/mol/K. Thermal stability of lipase was found to be at 20˚C. Lipase activity retained up to 3 h of incubation with 10 mM and 20 mM of CaCl 2 in starting buffer. This shows that Calcium has enhancing property from the denaturation of enzyme. Michaelis-Menten constant (K m) of lipase from Indian major carp, catla for the hydrolysis of pNPP was 6.695 mM. Turnover number (k cat) of lipase (catla) was 0.0022 s-1. Catalytic efficiency (k cat /K m) of lipase was 0.0003412 s-1 mM-1. SDS-PAGE of purified lipase (PF) revealed a homogenous single band with molecular mass of 70 kDa. Secondary structural arrangement of α helices and β strands of purified lipase results 48.51% and 9.74%, respectively using Circular Dichroism.

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Section: Sds-pagesupporting

confidence: 53%

Section: Physical Characterizationmentioning

confidence: 99%

Section: Kinetic Studymentioning

confidence: 99%

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Isolation, Purification and Characterization of Secondary Structure and Kinetic Study of Lipase from Indian Major Carp, Catla catla (Catla)

Tyagi¹

2014

Enz Eng

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“…For example, further addition of INS would inhibit the actions of GLU, stopping the lipolytic actions of lipase enzymes. Trout liver is known to possess a lipase which is activated by phosphorylation (Harmon et al 1991).…”

Section: Discussionmentioning

confidence: 99%

Insulin stimulates hepatic lipogenesis in rainbow trout, Oncorhynchus mykiss

Cowley

Sheridan

1993

Fish Physiol Biochem

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The effects of the pancreatic hormones, insulin and glucagon, on rates of lipid biosynthesis in liver removed from rainbow trout, Oncorhynchus mykiss, were evaluated in vitro. Livers were removed from animals fasted for 30-36h, cut into ca. 1 mm(3) pieces, and incubated in the presence of various concentrations of salmon insulin (sINS), bovine insulin (bINS), or a combination of BINS and bovine/porcine glucagon (GLU). Lipid synthesis was evaluated by total lipid concentration, (3)H2O incorporation into total lipid, and by fatty acid synthetase activity. Both mammalian and sINS tended to increase tissue total lipid concentration in hepatic tissue incubated for 5h. Insulin also stimulated (3)H2O incorporation into total lipid in a dose-dependent manner. Bovine INS (2 × 10(-6) M) stimulated de novo synthesis nearly 6-fold over control rates; sINS (2 × 10(-6) M) stimulated label incorporation more than 7-fold over control rates. Glucagon inhibited INS-stimulated (3)H2O incorporation; whereas, GLU alone had no effect on lipid synthesis in liver pieces incubated 5h. Lipid class analysis indicated that bINS significantly stimulated (3)H2O incorporation into phospholipids, fatty acids, and triacylglycerols. The greatest accumulation of label was in the triacylglycerol fraction, where incorporation was stimulated 17-fold over control levels. Hepatic enzymatic analysis indicated that bINS also significantly stimulated lipogenic enzyme activity 9-fold above control levels. These results indicate that INS is an important regulator of lipid synthesis in the liver of trout.

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“…The cells were resuspended in Hank's buffer and homogenized. Following centrifugation (16 000 g for 10 min at 14 8C), the supernatant was removed, separated, and subjected to 20% ammonium sulfate fractionation; the HSL-containing precipitate (Harmon et al 1991) was collected after ice incubation (30 min) and centrifugation (16 000 g for 15 min). HSL samples were resuspended in a buffer (25 mM Tris-HCl, pH 7.4) and subjected to SDS-PAGE.…”

Section: Hormone-modulated Phosphorylationmentioning

confidence: 99%

PKC and ERK mediate GH-stimulated lipolysis

Bergan

Kittilson

Sheridan

2013

Journal of Molecular Endocrinology

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GH regulates several physiological processes in vertebrates, including the promotion of growth, an anabolic process, and the mobilization of stored lipids, a catabolic process. In this study, we used hepatocytes isolated from rainbow trout (Oncorhynchus mykiss) as a model to examine the mechanism of GH action on lipolysis. GH stimulated lipolysis as measured by increased glycerol release in both a time-and a concentration-related manner. The promotion of lipolysis was accompanied by GH-stimulated phosphorylation of the lipolytic enzyme hormone-sensitive lipase (HSL). GH-stimulated lipolysis was also manifested by an increased expression of the two HSL-encoding mRNAs, HSL1 and HSL2. The signaling pathways that underlie GH-stimulated lipolysis were also studied. GH resulted in the activation of phospholipase C (PLC)/protein kinase C (PKC) and the MEK/ERK pathway, whereas JAK-STAT and the PI3K-Akt pathway were deactivated. The blockade of PLC/PKC and the MEK/ERK pathway inhibited GH-stimulated lipolysis and GH-stimulated phosphorylation of HSL as well as GH-stimulated HSL mRNA expression, whereas the blockade of JAK-STAT or the PI3K-Akt pathway had no effect on the activation of lipolysis or the expression of HSL stimulated by GH. These results indicate that GH promotes lipolysis by activating HSL and by enhancing the de novo expression of HSL mRNAs via the activation of PKC and ERK. These findings also suggest molecular mechanisms for activating the lipid catabolic actions of GH while simultaneously deactivating anabolic processes such as antilipolysis and the growth-promoting actions of GH.

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Purification and characterization of hepatic triacylglycerol lipase isolated from rainbow trout,Oncorhynchus mykiss

Cited by 20 publications

References 21 publications

Isolation, Purification and Characterization of Secondary Structure and Kinetic Study of Lipase from Indian Major Carp, Catla catla (Catla)

Isolation, Purification and Characterization of Secondary Structure and Kinetic Study of Lipase from Indian Major Carp, Catla catla (Catla)

Insulin stimulates hepatic lipogenesis in rainbow trout, Oncorhynchus mykiss

PKC and ERK mediate GH-stimulated lipolysis

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